Olefin Isomers of a Triazole Bisphosphonate Synergistically Inhibit Geranylgeranyl Diphosphate Synthase

Abstract

The isoprenoid donor for protein geranylgeranylation reactions, geranylgeranyl diphosphate (GGDP), is the product of the enzyme GGDP synthase (GGDPS) that condenses farnesyl diphosphate (FDP) and isopentenyl pyrophosphate. GGDPS inhibition is of interest from a therapeutic perspective for multiple myeloma because we have shown that targeting Rab GTPase geranylgeranylation impairs monoclonal protein trafficking, leading to endoplasmic reticulum stress and apoptosis. We reported a series of triazole bisphosphonate GGDPS inhibitors, of which the most potent was a 3:1 mixture of homogeranyl (HG) and homoneryl (HN) isomers. Here we determined the activity of the individual olefin isomers. Enzymatic and cellular assays revealed that although HN is approximately threefold more potent than HG, HN is not more potent than the original mixture. Studies in which cells were treated with varying concentrations of each isomer alone and in different combinations revealed that the two isomers potentiate the induced-inhibition of protein geranylgeranylation when used in a 3:1 HG:HN combination. A synergistic interaction was observed between the two isomers in the GGDPS enzyme assay. These results suggested that the two isomers bind simultaneously to the enzyme but within different domains. Computational modeling studies revealed that HN is preferred at the FDP site, that HG is preferred at the GGDP site, and that both isomers may bind to the enzyme simultaneously. These studies are the first to report a set of olefin isomers that synergistically inhibit GGDPS, thus establishing a new paradigm for the future development of GGDPS inhibitors.

Bishomoisoprenoid triazole bisphosphonates as inhibitors of geranylgeranyl diphosphate synthase

“…With respect to the olefin stereochemistry, these results are consistent with the previously described geranyl/neryl and homogeranyl/homoneryl sets of triazole bisphosphonates. 9,12 Interestingly, however, while the results of all three assays were concordant for 17a and 17c , 17b appeared to more potently disrupt Rap1a geranylgeranylation (Figure 3A) than Rab geranylgeranylation (Figure 2B–C). …”

“…We already have demonstrated that the Rap1a and ELISA assays give concordant results for GGDPS inhibitors. 9,10,12,29 The HMG-CoA reductase inhibitor lovastatin, which inhibits the synthesis of mevalonate and therefore depletes cells of all isoprenoids downstream of mevalonate, was used as a positive control in these studies. 6 Of the three compounds examined, the bishomoneryl compound 17c most potently disrupted protein geranylgeranylation, with activity seen at a concentration as low as 0.5 μM.…”

“…Because we had previously demonstrated that the combination of the homogeranyl and homoneryl triazole bisphosphonates synergistically inhibited GGDPS, 12 we investigated whether the bishomo isomers behaved in a similar manner. The combination of the two isomers, either in a 1:1 or 3:1 (bishomogeranyl:bishomoneryl) mixture, did enhance GGDPS inhibition compared with either isomer alone (Supplementary Figure 1), but was not as strongly synergistic as the homoisomers.12 Similar studies performed in cell culture revealed that the combination of the two isomers did not result in a greater accumulation of unmodified Rap1a (Supplementary Figure 2).…”

“…11 These studies revealed that while the homoneryl isomer ( 6 ) was more potent than the homogeranyl isomer ( 4 ), when used in combination the two isomers exhibited synergistic inhibitory effects on the target enzyme. 12 Modeling studies suggested that the homoneryl isomer ( 6 ) preferentially binds to the FDP site while the homogeranyl isomer ( 4 ) preferentially binds to the GGDP site, thus establishing a new paradigm for the development of GGDPS inhibitors. 12 We were therefore interested in determining whether further extension of the tether between the triazole and the internal olefin would affect GGDPS inhibitory activity.…”

“…12 Modeling studies suggested that the homoneryl isomer ( 6 ) preferentially binds to the FDP site while the homogeranyl isomer ( 4 ) preferentially binds to the GGDP site, thus establishing a new paradigm for the development of GGDPS inhibitors. 12 We were therefore interested in determining whether further extension of the tether between the triazole and the internal olefin would affect GGDPS inhibitory activity. To begin to address this question we report the synthesis and biological activity of three new compounds that can be viewed as the bishomoprenyl, bishomogeranyl, and bishomoneryl triazoles.…”